Oligonucleotides and oligonucleotide analogs (hereinafter referred to as "oligomeric compounds") of known sequences are utilized in a wide variety of chemical and biological applications, including PCR (polymerase chain reaction) and molecular cloning, as well as in the diagnosis and treatment of diseases (see, for example, Antisense Research and Applications, Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993). It is often desirable to detect, isolate and/or quantitate a specific, desired oligomeric compound present in a complex mixture which may also include other closely related oligomeric compounds. Such other closely related oligomeric compounds may be less than full length as compared to the oligomeric compound of interest but otherwise have the same sequence, or may differ from a desired sequence by one or only a few bases. This is especially important in biological samples, where the presence or absence of specific known nucleotide sequences can be indicative of the presence or absence of an added oligonucleotide agent or, alternatively, a disease state. It is also important in assays to determine the effect of particular enzymes on selected oligomeric compounds, especially in cases where enzymes possess nuclease activity. The foregoing considerations are also important in the manufacture of oligonucleotides, for example, to characterize the purity of the product.
Techniques for the detection and quantification of oligomeric compounds are known. However, samples of interest often do not contain sufficient concentrations of oligonucleotides to permit detection by techniques such as ultraviolet (UV) spectroscopy. Additionally, samples often contain other absorbing species that prohibit detection of the species of interest. Other analytic techniques may lack specificity for a particular nucleic acid sequence, or require excessive sample preparation or analysis times.
The use of electrophoretic techniques to separate oligonucleotide species is documented in the literature. One such technique is capillary electrophoresis (CE), which employs relatively long, thin capillary columns for the separation of oligonucleotides. See generally, Capillary Electrophoresis Theory and Practice, P. Grossman and L. Colburn, eds. Academic Press, New York (1992), and Janson Ryden, Protein Purification, Ch. 17, VCH Publishers, New York, N.Y. CE affords several advantages over conventional electrophoretic techniques such as polyacrylamide gel electrophoresis (PAGE). for example, because CE is performed in very small diameter tubing (typically 50-100 .mu.m i.d.), electric fields 10 to 100 fold greater than those used in conventional electrophoretic systems can be applied because of reduced Joule heating. This affords very high run speeds and improved resolution. Also, CE lends itself to on-column detection means including ultraviolet (UV) spectroscopy, amperometric measurement, conductivity measurement, laser-induced fluorescence detection (LIF) or thermooptical detection. Additionally, CE can be performed with or without a gel medium in the capillary. The use of electrophoretic techniques to separate oligonucleotide species using gels such as polyacrylamide gel is referred to as capillary gel electrophoresis (CGE).
There have been several reports of the use of CE in the detection of DNA species, such as in the high speed sequencing of DNA. For example, Luckey et al., Nucleic Acids Research, 1990, 18, 4417-4421, describes a CE instrument developed for automated DNA sequencing in which products are detected via the fluorescence of an intercalating dye.
CE analysis of PCR amplified DNA has been reported using non-gel sieving buffers and fluorescent intercalating dyes. The identification of DNA molecules by pre-column hybridization followed by capillary electrophoresis with on-line fluorescence detection has been described (Chen et al., Journal of Chromatography, 1991, 559, 295-305).
CGE has been used to separate peptide nucleic acid (PNA)-oligonucleotide heteroduplexes from free single-strand oligonucleotide and single strand peptide nucleic acid (Rose et al., Anal. Biochem., 1993, 65, 3545-3549). PNAs are capable of hybridization to complementary DNA or RNA sequences to form hybridized moieties which are more stable (i.e., which have higher binding affinities and higher melting temperatures) than corresponding "natural" duplexes. See Antisense Research and Applications, Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993.
Central to the development of antisense therapeutics possessing useful pharmacological activity is the issue of nuclease resistance. Nucleases are enzymes which degrade nuclei acids into smaller pieces. For example, endonucleases cleave nucleic acids at internal sites (the phosphodiester bonds) in the nucleotide sequence. Exonucleases, on the other hand, cleave nucleotides sequentially from the free ends of linear nucleic acids. It has been well documented in the literature that short, unmodified oligonucleotides are inherently unstable in biological systems (as first reported by Wickstrom, E. J. Biochem. Biophys. Methods, 1986, 13, 97). This was demonstrated by showing that the half life of a short, unmodified oligonucleotide in fetal calf serum was less than half of an hour.
Nuclease stability assays are one of several screens typically performed to evaluate the usefulness of new antisense compounds. For example, nuclease stability assays are used to determine if the stability of an oligonucleotide analog to nucleases is greater than that of unmodified oligonucleotide. Nuclease stability assays are one of several screens done in an attempt to evaluate the usefulness of new antisense chemistries.
Because of the possibility that endogenous nuclease activity can degrade an oligonucleotide therapeutic before it can exert its beneficial effects, oligomeric compounds which are highly susceptible to nuclease activity are, in most situations, less desirable than those which are nuclease resistant. However, it is often difficult to determine the nuclease stability of an oligomeric compound in a solution containing many components using present methods.
Therefore, there exists a long-felt need for methods of detecting and quantifying products of nuclease digestions of target oligomeric compounds to ascertain determine the nuclease stability of the compounds (i.e., "determining the nuclease stability") the nuclease stability of oligomeric compounds that overcome the limitations posed by present methods. The present invention is directed to these, as well as other, important ends.